The ligand-gated potassium channels KefC and KefB of <i>Escherichia coli </i>are critical components in protecting cells from toxic electrophiles.  Potassium efflux through these channels is coupled to a decrease in cytoplasmic pH which in turn reduces the damage to DNA by electrophiles.  KefC and KefB are both inhibited by cytoplasmic glutathione and activated by glutathione adducts, such as ESG, formed by conjugation of glutathione with electrophilic compounds. Robust membrane purification protocols were developed to isolate both the wild type full-length KefC and KefB and the mutants required for biophysical analysis.  <i>In vivo </i>K⁺ measurements were performed to ensure that all of the constructs used were fully functional.  Structural and functional analysis used electron paramagnetic resonance (EPR) and stead state emission fluorescence measurements <i>in vivo</i>, on wild type and mutated full-length proteins to elucidate the gating mechanism and test the model generated from crystallographic data.  In particular, EPR spectroscopy combined with site-directed spin labelling revealed a substantial conformational change and thus provided the first insight into coupling between sensing and gating.  Steady state fluorescence spectroscopy was used to precisely measure binding affinities for both activating and inhibitory ligands and characterise nucleotide binding to KefC.  Finally, a variety of chemically diverse glutathione adducts was tested on KefC <i>in vitro </i>to elucidate the mechanism by which these ligands initiate K⁺ flux through the associated transmembrane domain.

The cytochrome o and cytochrome d oxidase complexes provide twin termini for the branched respiratory chain of aerobically grown Escherichia coli. Combined use of mutant strains, modulated growth conditions and high resolution analytical techniques enabled cytochromes to be resolved, identified and partially characterized. The cytochrome complement of everted membrane vesicles and detergent extracts fractionated by liquid chromatography is more complex than previously recognised. Multiple type-b cytochromes were resolved by potentiometry and by high resolution spectrophotometry in membrane vesicles from mutant strains lacking the cytochrome d oxidase complex and grown under conditions minimising respiratory chain diversity. Cytochrome o was identified with Em = +235 mV (vs. NHE) as were low potential cytochromes associated with dehydrogenases. Spectrally distinct components of the cytochrome d complex yielded Em values of +125 mV (cytochrome 6595) and +187 mV (cytochrome d). The latter displayed atypical redox behaviour with extreme hysteresis during potentiometric titrations. Several cytochromes b₅₅₆ displaying single, symmetrical redox α-bands at 77 K were resolved from detergent extracts of vesicles. Mutant strains identified one with Mr = 52500 (gel filtration) and Em = +20 mV as the sdhC gene product, a component of succinate dehydrogenase. DL-lactate induced another while a hydroperoxidase, Mr = 386000 (gel filtration) with twin Em values of -2mV and -121 mV and a split Soret absorption band at 77 K (λ[symbol omitted]max= 426.0 nm + 434.0 nm) was produced under limited oxygen tension. The Triton-solubilized and purified cytochrome 0 complex exhibited Mr = 516000 (gel filtration) with five component peptides of Mr= 55000, 32000, 31000, 21000 and 16000 (SDS-PAGE). It displayed mid-point potentials of -58 mV, +127 mV and +260 mV and three a-absorption maxima at 77 K : 554.5 nm, 557.0 nm and 563.5 nm. These components were reduced equivalently during poised-potential low temperature spectrophotometric analyses. Carbon monoxide binding changed the complex's redox α-absorption spectrum minimally but shifted the high potential Em to approximately +420 mV. Quinone analogues inhibited both reduction and reoxidation of the complex. Cytochrome o complex prepared from cloned sources contained a significantly greater proportion of the component with mid range electrochemical potential absorbing at 554.0 nm. These results are discussed in relation to possible structures of the complex, its respiratory interactions and the identity of cytochrome o itself. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate